Strains of Escherichia coli which produce isoleucine or valine and a method for their production

ABSTRACT

A method for constructing microorganism strains which produce amino acids comprising: combining in one bacterial genome (a) a mutation affecting the aminoacyl-tRNA synthetase corresponding to the selected amino acid, conferring cells auxotrophy which cannot be fully suppressed by addition of usual concentration this amino acid into the culture medium and (b) a mutation which destroys the negative regulation of selected amino acid biosynthesis to yield a strain capable of increased production of the selected amino acid. 
     Strains producing isoleucine and valine. 
     Methods to produce isoleucine and valine by culturing those strains.

This is a division of application Ser. No. 08/116,601 filed on Sep. 7,1993, now U.S. Pat. No. 5,534,421, which is a division of Ser. No.07/707,616, filed on May 30, 1991, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the microbiological industry and, morespecifically, it relates to a method for preparing strains which produceamino acids.

DISCUSSION OF THE BACKGROUND

Amino acids produced by microorganisms find extensive use as feedstuffand food additives in the agriculture and food industry, as componentsof various nutrient mixtures for medical purposes and as reagents in thechemical and pharmaceutical industries.

Known in the art are methods for preparing strains which produce aminoacids such as L-lysine, L-threonine, L-isoleucine, L-valine and the likeby using various mutagens. The resulting mutant strains ofmicroorganisms have specific genetically preconditioned defects inregulating metabolism and, due to such defects, they evolve into thenutrient medium, or produce specific amino acids. The required strainsof microorganisms are produced by conventional methods based on theparticular nutritive demand of a mutant (auxotrophy) or on resistance ofa mutant to one or another structural analogue of an amino acidinhibiting the growth of the parental strain.

Amino acid production by known auxotrophic strains results from blockingthe formation of a by-product or a coinhibitor amino acid, whichparticipate in the negative control of the amino acid biosynthesis.Known are pantothenate auxotrophs of Escherichia coli, producing valine(Maas, W. K., Vogel, H. J., J. Bacteriol., v. 65, p. 388, 1953),homoserine-requiring strains of Corynebacterium glutamicumandBrevibacterium flavum, producing lysine (Nakayama et al., J. Gen. Appl.Microbiol., v. 7, p. 41, 1961), isoleucine-, threonine-, orhomoserine-requiring mutants of Arthrobacter paraffineus andCorynebacterium hydrocarboclastus producing valine (U.S. Pat. No.3,700,556).

Amino acid production by mutant strains resistant to structuralanalogues of amino acids results from destroying the negative regulationof the amino acid biosynthesis, i.e., feed-back inhibition of the keyenzyme activity or repression of the corresponding enzyme's formation bythe end products. Known are the S-2-aminoethyl-L-cysteine resistantmutant of genus Brevibacterium producing lysine (sano, K., Shiio, J.Gen. Appl. Microbiol., v. 16., p. 373, 1970, Shiio et al., J. Biochem.,v. 68, p. 701, 1970), the amino-hydroxyvaleric acid (AHV) resistantmutant of Escherichia coli producing threonine (Shiio, I., Nakamoris,Agr. Biol. Chem., v. 33, p. 1152, 1969), AHV resistant mutant of theBrevibacterium producing threonine and the mutant of microorganismsbelonging to the genus Brevibacterium or Corynebacterium, resistant toamino-hydroxyvaleric acid producing isoleucine (U.S. Pat. No.3,767,529), mutant strains of the genera Brevibacterium andCorynebacterium having resistance to 2-thiasolalanine which producevaline (U.S. Pat. No. 3,893,888), mutant strains of Serratia marcescensresistant to isoleucine hydroxamate producing isoleucine (Kisumi etal,J. Bacteriol., v. 110, p. 761, 1972).

Amino acid-producing strains having a resistance to an amino acidanalogue together with a nutrient requirement which increases theirproductivity are also known (U.S. Pat. No. 3,893,888). Such amino acidproducers remain auxotrophic and can grow only on media containingspecific additives.

Known in the art is also a method for preparing bacterial strains thatproduce amino acids which is based on the isolation of chromosome DNAfragments of a donor bacterium containing genes controlling thesynthesis of a'selected amino acid, combining them with a multicopyplasmid DNA molecule by in vitro manipulation, and transforming arecipient strain with a hybrid DNA molecule to yield a bacterial strainpossessing increased productivity (U.S. Pat. Nos. 4,278,765; 4,391,907).According to this method chromosomal DNA fragments are isolated from astrain a having a mutation which destroys the negative regulation ofselected amino acid biosynthesis. The recipient strain may be aspecially constructed strain or it may be the donor strain.

However no effective Escherichia coli strains producing isoleucine orvaline have been obtained by this method. Therefore a need continues toexist for the development of a novel method for preparing amino acidproducing strains.

Hitherto unknown are strains characterized by increasing production ofan amino acid due to mutation in genes coding for the correspondingaminoacyl-tRNA synthetase. The aminoacyl-tRNA synthetases, or activatingenzymes, are particularly crucial elements in the route leading fromamino acids to proteins. These enzymes catalyze the formation ofactivated amino acids, that is their attachment to one or more specifictRNA. In most cases there is but one aminoacyl-tRNA synthetase for eachamino acid. So, only conditionally expressed mutations may be obtained.

Mutants with altered aminoacyl-tRNA synthetases were described inseveral papers:

1. Roth, J. R., and Ames, B. N., J. Mol. Biol., v. 22, p. 325, 1966.

2. Neidllardt, F. C., Bacteriol. Rev., v. 30, p. 701, 1966.

3. Folk, W. R., and Berg, P., J. Bacteriol., v. 102, p. 193, 1970.

4. Iaccarino, M., and Berg, P., J. Bacteriol., v. 105, p. 527, 1971.

5. Johnson, E. M. et al, J. Bacteriol., v. 129, p. 66, 1979.

Some aminoacyl-tRNA synthetase mutations manifest themselves asauxotrophy. This is an unusual auxotrophy, because the defect is not inthe formation of an amino acid, but in its utilization for proteinsynthesis. Also it was communicated, that phenotypic suppression of suchauxotrophic mutations may arise as a result of additional mutationswhich increase the biosynthesis and intracellular concentration of thecorresponding amino acid. However, amino acid producing strainsconstructed on the basis of auxotrophic aminoacyl-tRNA synthetasemutations and methods for their production were not known. Also, therole of such mutations in overproduction of amino acids by producerstrains was not established.

SUMMARY OF THE INVENTION

It is an object of the present invention to use aminoacyl-tRNAsynthetase mutations for preparing strains possessing enhancedcapability of producing amino acids without additional growth factors,more specifically, strains of Escherichia coli producing isoleucine andvaline.

These and other objects of the invention have been attained bydevelopment of a method for preparing strains which produce enhancedlevels of amino acids due to a mutation affecting aminoacyl-tRNAsynthetase conferring cells auxotrophy which can be compensated onlypartially by addition of the selected amino acid into the culturemedium. This is combined with mutations which destroy the negativeregulation of the amino acid biosynthesis to give a strain capable ofincreased productivity of the selected amino acid.

DETAILED DESCRIPTION OF THE INVENTION

Mutations affecting aminoacyl-tRNA synthetases which manifest themselvesas auxotrophy have many times decreased affinity (increased the Km) forthe corresponding amino acids (3-4). Therefore the formation of theactivated amino acids (the charging of the corresponding tRNA) in theircells is decreased. Under this condition the transcription of thestructural genes (operons) involved in the biosynthesis of these aminoacid may be markedly increased, if this expression is controlled byattenuation. This regulatory mechanism exists in many amino acid operonsof different bacterial species (Kolter, R., Yanofsky, C., Ann. Rev.Genet., v. 16, p. 113, 1982; Matsui, K. et al, Nucleic Acids Res., v.14, p. 10113, 1986; Shimotsu, H. et al, J. Bacteriol., v. 166, p. 461,1986; Kuroda, M. I. et al, J. Bacteriol., v. 167, p. 792, 1086.).

Besides, the decreased tRNA charging elevates the intracellularconcentration of guanosine tetraphosphate (ppGpp), which is known toactivate initiation of transcription of amino acid operons (Stephens, J.C. et al, Proc. Nat. Acad. Sci. USA, v. 72, p. 389, 1975; Cashel, M.,and Rudd, K. E. In: "Escherichia coli and Salmonella typhimurium:Cellular and Molecular Biology (vol. 12, Neidhardt, F. C., ed.), p.1410, ASM, Washington, 1987). Taken together these factors create thepossibility for overproduction of the corresponding amino acid. Thispossibility can be realized if the negative regulation of the amino acidbiosynthesis is eliminated.

To obtain the high level of amino acid operons expression, which isnecessary for effective amino acid production, only those aminoacyl-tRNAsynthetase mutations must be used which cannot be fully suppressed(compensated) by addition of the corresponding amino acid into themedium. These mutations may be produced by conventional process formutation induction such as by treating the prototrophic parent strainwith N-methyl-N'-nitro-N-nitrosoguanidine. The screening procedureshould include selection of auxotrophic mutants, which can grow onlyslowly on media containing high concentrations (3-10 mg/ml) of thecorresponding amine acid, and cannot grow without it. This procedurepermits one, to obtain just the motants with impaired aminoacyl-tRNAsynthetases, because the auxotrophy caused by blocks in the amino acidbiosynthetic pathway can be completely compensated by addition of lowconcentrations (0.005-0.05 mg/ml) of the amino acid into the medium.

On the basis of the mutant strains additional mutations, those withincreased intracellular concentration of the corresponding amino acidmay be obtained by selecting (pseudo)revertants, which can grow on mediacontaining no amino acid or containing low concentrations of the aminoacid. By using this procedure different mutations in the genes involvedin amino acid overproduction may be induced, and step-by-step selectionleading to increased amino acid productivity may be performed.Maintenance of the defect in the aminoacyl-tRNA synthetase in revertantsmust have positive effect on their productivity. Thus, effective aminoacid producing strains may be prepared.

In addition, these types of mutations affecting aminoacyl-tRNAsynthetase may be introduced into the chromosome of amino acid producersobtained by known procedures, including selection foranalogue-resistance, by transformation, transduction, conjugation orprotoplast fusion to increase their productivity. Also, theabove-described mutations affecting aminoacyl-tRNA synthetases may beintroduced into the chromosome of recipient strains by recombinant DNAtechniques.

Using the method of the present invention, mutant strains wereconstructed which produce valine and isoleucine in higher yields thanhas been achieved with previously known methods using artificial mutantsof Escherichia.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are givenfor illustration and not intended to be limiting.

EXAMPLE 1

1. Selection of mutants requiring high isoleucine concentration fortheir growth.

Escherichia coli K12 W3350 was treated withN-methyl-N'-nitro-N-nitrosoguanidine and cultured with shaking in thepresence of 10 mg/ml of L-isoleucine at 37° C. for 24 hours. The cellswere then washed, resuspended in minimal medium supplemented withglucose (0.2%) and washed at 37° C. When a doubling of optical density(A590) was obtained, 2000 units of penicillin per ml was added, andafter 3 hours incubation at 37° C. the cells were washed and spread onminimal agar plates supplemented with 0.2% glucose and 10 mg/mlL-isoleucine. Isoleucine auxotrophs which can grow only in the presenceof high isoleucine concentration in the medium were selected using thestandard procedure. Three independent mutants have thus been isolated:leS2, IleS17, and-IleS32.

Table 1 shows the optical densities (A590) of the 18 hour cultures ofthe isoleucine-requiring mutants grown on minimal medium supplementedwith different isoleucine concentrations.

                  TABLE 1    ______________________________________             isoleucine (mg/ml)    Strain   0.00   0.01     0.05 1.0    3.0  5.0             Optical density (A590)    IleS 2   0.001  0.18     0.33 1.10   1.30 1.60    IleS 17  0.001  0.013    0.04 0.11   0.41 0.76    IleS 32  0.001  0.14     0.29 1.00   2.00 2.00    VL 330   0.001  0.93     1.70 1.60   1.40 1.40    W 3350   1.50   1.70     2.00 2.00   2.00 2.00    ______________________________________

The regular isoleucine auxotroph VL 330 (ilvA) reaches the maximum OD at0.05 mg/ml of isoleucine in a growth medium. The mutants IleS2, IleS17and IleS32 require much higher isoleucine concentration for theirgrowth. Furthermore, the mutant IleS17 grew slowly even in the presenceof 5 mg/ml of isoleucine in the medium.

Genetic data has shown that mutations conferring isoleucine auxotrophyto the IleS strains were mapping on the Escherichia coli chromosomebetween the threonine and leucine operons where the gene coding forisoleucyl-tRNA synthetase is known to be located. Accordingly,isoleucyl-tRNA synthetase activities in the IleS2 and IleS17 strainsmeasured by a known method were 43% and 37% of the respective activityin the parental strain Escherichia coli W 3350.

2. Selection of the mutants requiring high valine concentration fortheir growth.

Escherichia coli VL1502 (valR, pyrB::Tn5), W 3350 derivative was treatedwith N-methyl-N'-nitro-N-nitrosoquanidine and cultivated with aerationin the presence of 10 mg/ml of L-valine at 37° C. for 30 hours. Then,after two rounds of the penicillin enrichment procedure described above,valine auxotrophs were selected. About 20% of colonies tested werevaline-requiring. Nine independent ValS mutants have thus been isolated.

Table 2 shows the optical densities (A590) of 20 hour cultures of someValS mutants grown on minimal medium supplemented with valine indifferent concentrations.

                  TABLE 2    ______________________________________    valine (mg/ml)    Strain  0.00   0.05   0.2  0.5  1.0  2.0  5.0 7.0 10.0    Optical density (A590)    ValS 52 0.005  0.01   0.02 0.03 0.07 0.16 1.5 1.8 1.4    ValS 67 0.01   0.04   0.07 0.21 0.50 1.8  2.2 1.9 1.5    VAlS 91 0.02   0.03   0.04 0.08 0.15 0.58 1.9 1.8 1.45    C600 ilvc*            0.01   2.0    2.2  2.3  2.2  2.5  2.3 2.2 2.2    ______________________________________     *For this strain 0.1 mg/ml of isoleucine was added together with indicate     valine concentrations

All the mutations conferring valine-requirement to ValS mutants werecotransduced with high frequency by phage P1 with pyrB::Tn5 marker(82-96%). This result indicates that they are located in the same regionwhere known valS mutations were mapped. Activity of valyl-tRNAsynthetase was measured by a known method in several ValS mutants andfound to be decreased. In ValS52 mutant it was only 16% of therespective activity in the parental strain.

Thus, by using the described procedure mutants impaired inaminoacyl-tRNA synthetase activities can easily be selected.

3. Measuring of guanosinetetraphosphate level in IleS 17 mutant.

During the balanced growth IleS and ValS mutants may have decreasedlevels of charged isoleucyl-tRNA or valyl-tRNA due to reduced activityof the corresponding aminoacyl-tRNA synthetase. This must lead to theincreased formation of guanosinetetraphosphate (ppGpp). So intracellularppGpp concentration was measured by using a known method in the IleS17mutant and in the parental strain W3350 grown in minimal mediumsupplemented with isoleucine.

                  TABLE 3    ______________________________________               Isoleucine concentration                              ppGpp    Strain     in medium (mg/ml)                              pmole/A450    ______________________________________    W3350      2              35    IleS17     2              140               3              105               5              55               10             30    ______________________________________

The results presented in Table 3 show that under the same growthconditions the ppGpp concentration in IleS17 mutant is four times higherthan in the strain W3350. The ppGpp level was dependent on isoleucineconcentration in growth medium. The higher was the isoleucineconcentration the lower was the level of ppGpp in IleS17 cells.

4. Determination of threonine deaminase activity in IleS mutants.

Isoleucine and valine production by Escherichia coli strains must dependon the level of ilv-operon expression. To determine the effect of ileSmutations on expression of the ilvGMEDA operon the threonine deaminaseactivity was measured by a known method in IleS mutants grown on mediumsupplemented with isoleucine in low (0.01 or 0.05 mg/ml) and high (5.2mg/ml) concentrations (Table 4).

                  TABLE 4    ______________________________________             Isoleucine concentration                            Relative threonine    Strain   in medium (mg/ml)                            deaminase activity (%)    ______________________________________    IleS 2   0.01           757             5.20            87    IleS 17  0.05           658             5.20           366    IleS 32  0.01           682             5.20           122    W3350    0.00           100             5.20            26    ______________________________________

The results presented in Table 4 show that the enzyme activities in theIleS strains were higher than in the parental strain W3350 underdifferent growth conditions. It can also be seen that in the presence of5.20 mg/ml of isoleucine in medium the highest threonine deaminaseactivity was in the IleS17 mutant (14 times higher than in W3350).

EXAMPLE 2

1. Selection of revertants of IleS strains which produce isoleucine.

The strains IleS2, IleS17 and IleS32 were treated withN-methyl-N'nitro-N-nitrosoguanidine, spread on plates containing minimalmedium supplemented with 0.2% glucose or 0.2% glucose and 3 mg/mlthreonine and cultured for 48-72 hours. The revertants which appearedwere tested for their ability to excrete isoleucine into a medium and tofeed the lawn of isoleucine-requiring mutant IlvA (syntrophism test,Table 5).

                  TABLE 5    ______________________________________                Number of   Number of    Parent strain                revertants tested                            isoleucine excretors    ______________________________________    IleS 2      50          14    IleS 17     100         25    IleS 32     75          24    ______________________________________

Thus, using IleS mutants isoleucine producing strains can easily beobtained.

Threonine deaminase activity and its inhibition by the end product wasdetermined in revertants excreting isoleucine. In most of them theenzyme was not inhibited by 1 mM of isoleucine. Furthermore, in therevertant Rev 7434 (derivative of IleS2) threonine deminase wascompletely insensitive to inhibition by the end product. Genetic andnucleotide sequence analysis of Rev 7434 has shown that the mutationconferring resistance to the inhibition was in the ilvA gene coding forthreonine deaminase (ilvA7434 mutation).

2. Construction of the isoleucine producing strain containing valR,ilvA7434, and ileS17 mutations.

The strain Rev7434, as well as other Escherichia coli K12 strains,contains a mutation in the ilvG gene which decreases the ilvGMEDA operonexpression. To eliminate this defect a spontaneous valine-resistantmutant of this strain was obtained by plating it on agar minimal mediumsupplemented with glucose (0.2%) and L-valine (1 mg/ml). Thus, ValRstrain VL1886 was obtained which contains a mutation conferringvaline-resistance located in the ilvGMEDA operon. Phage P1 grown on thestrain VL1886 was used for transduction of the strain VL334 (thrC ilvA).On minimal medium supplemented with glucose (0.2%) and threonine (0.05mg/ml) Ils+ transductants were selected. Over 90% of them werevaline-resistant and had threonine deaminase insensitive to inhibitionby isoleucine. Thus the strain VL1887 (thrC ilvA7434 valR) was obtained.Then phage P1 grown on IleS17 was used for transduction of the strainVL1887. On minimal medium supplemented with glucose Thr+ transductantswere selected. About 50% of them grew slowly, and their growth wasstimulated by isoleucine. These were transductants which received ileS17mutation.. Thus the strain VL1892 was constructed which containssimultaneously valR, ilvA7434, and ileS17 mutations. This strain hadelevated levels of isoleucine-insensitive threonine deaminase which wasfrom 6 to 10 times higher than in Rev7434. The synthrophism test showedthat the strain VL 1892 produces isoleucine.

3. Construction of the isoleucine producing strain containingilv-operons on low copy number plasmid.

The strain AB 1206 harbors a low copy number plasmid F'14, whichcontains a chromosome fragment with ilv-operons and a correspondingchromosome deletion. Phage P1 grown on VL1886 was used for transductionbf AB 1206. The transductants were selected on minimal mediumsupplemented with-glucose (0.2%) and valine (1 mg/ml). Thus the stain KX139 was obtained in which the F'14 plasmid contains the valR and ilv7434mutations established by measuring threonine deaminase activity by aknown method. The plasmid can be transferred by conjugation in differentEscherichia coli strains, more specially, in IleS17. However in RecA+strains it might be unstable because of the frequent integration intothe chromosome. Therefore the recA mutation was introduced into thechromosome of IleS17 strain by using the known conjugation procedurefrom the strain NK6659 (Hfr KL16 srl::Tn10 recA). Recombinants wereobtained on L-broth agar medium supplemented with tetracycline (0.01mg/ml) and among them a UV-sensitive IleS strain was selected. Thus thestrain KX140 having genotype ileS17 recA was prepared. The plasmid F'14(valR ilv7434) was introduced into the cells of KX140 by conjugationgiving the strain KX 141. The syntrophism test showed that this strainproduces isoleucine.

4. Production of L-isoleucine by the novel L-isoleucine-producingstrains.

The strains of Escherichia coli VL 1892 and KX141 were inoculated byloop from the slant of an agarized M9 medium into Erlenmeyer flasks eachcontaining 50 ml of L-broth (containing 10 g/l peptone, 5 g/l yeastextract, 1 g/l glucose and 5 g/l NaCl, pH 7.2). After inoculation theflasks were placed on a circular shaker (200 r.p.m.) and incubated forhours at the temperature 37° C. The thus prepared material was used as aseed culture.

A main culture medium, containing 30 g/l glucose, 5 g/l ammoniumsulfate, 2 g/l K₂ HPO₄, 0.4 g/l MgSO₄ ·7H₂ O, 0.02 g/l FeSO₄ ·7H₂ O,0.02 g/l MnSO₄ ·5H₂ O, 2 g/l yeast autolysate, 1 g/l L-threonine wasprepared. 300 ml of the medium was placed in a 0.5 l jar-fermentor andsterilized at 121° C. for 15 min. The medium was inoculated with 30 mlof the seed culture obtained above and cultivated with stirring at 900r.p.m. and introducing 1:1 volume of air per minute. The pH of themedium was maintained automatically around 7.2 by feeding, on pH-monitorsignal, a mixture containing in ratio 10:7:1 of solution of threonine,60% solution of glucose, and 25% solution of ammonia.

The fermentation was performed for 46 hours. By the end of fermentationthe culture medium with VL1892 contained 8-13 g/l of isoleucine, and theculture medium with KX 141 contained 11-17.8 g/l of isoleucine. Thestrain KX141 has been deposited in the All-Union Collection ofIndustrial Microorganisms in VNIIGENETIKA(USSR) as VKPM B-4781.

EXAMPLE 3

1. Selection of the revertants of IleS32 ValR strain which produceL-valine.

To obtain L-valine producers, the strain harboring both ileS and valR(ilvG) mutations was constructed. The valR (ilvG) mutation restores theactivity of the valine-resistant AHAS II--the key enzyme in valinebiosynthetic pathway, which is defective in the strains of E. coli K₁₂.By sequential phage Pl-mediated transduction mutation valR from thestrain C600 valR (resistant to 1 mg/ml of valine) and mutation ileS32from the strain IleS32 were introduced into the chromosome of the strainVL334 (thrC ilvA). On the plates of agarized minimal medium M9,containing glucose (0.2%) and valine (2 mg/ml), ValR transductants wereselected and on the plates of this medium containing isoleucine (2mg/ml) Thr+ IleS transductants were obtained.

Thus the strain IleS32 valR was constructed. Then the revertants of thisstrain were selected by plating it on M9 medium containing noisoleucine. The colonies which appeared were tested for their ability tofeed the lawn of isoleucine and valine requiring E. coli strain onminimal medium M9 containing isoleucine (0.05 mg/ml). Thus valineexcreting strains Rev835, Rev 839 and Rev874 were obtained.

By using phage Pl-mediated transduction it was found that all of therevertants contained ileS mutation which can be transferred in differentEscherichia coli strains.

2. Production of L-valine by the (pseudo)revertants.

The ability of the strains obtained to produce L-valine was tested byfermentation in tubes. The fermentation medium contained 50 g/l glucose,10 g/l ammonium sulfate, 1 g/l K₂ HPO₄, 0.4 g/l MgSO₄ ·4H₂ O, 0.02 g/lFeSO₄ ·7H₂ O, 0.02 g/l MnSO₄ ·5H₂ O, and 20 g/l CaCO₃ (separatelysterilized) and the pH was adjusted to 7.2.

Three ml batches of the fermentation medium were placed in 20 mm tubesinoculated with one loopful inoculum of the test strain cells, andcultivation was carried out at 37° for 46 hours.

The amount of L-valine in the supernatants of the fermentation broth isshown in Table 6.

                  TABLE 6    ______________________________________                  L-valine produced    Strained tested                  (g/l)    ______________________________________    Rev 835       4.3    Rev 839       1.2    Rev 874       5.6    ______________________________________

3. Construction of the effective L-valine producer.

To obtain a more effective valine producer and to find out the role ofileS mutations on valine production, isogenic IleS+ and IleS- wereconstructed on the basis of Rev 874. Phage P1, cultured on the cells ofdonor strain NK 6066 (thr::Tn9) was used for transduction of thr:Tn9into the chromosome of the Rev 874. The transductants were selected onL-broth agar plates, containing chloramphenicol (10 ug/ml). Among themthreonine-requiring strain VL 1966, fast-growing on minimal mediumcontaining no isoleucine (IleS+), was obtained. The cells of this strainwere transduced by phage P1 grown on IleS17. The transductants wereselected on M9 minimal medium containing glucose (0.2%) and isoleucine(2 mg/ml). Among Thr+ recombinants two strains, VL1968 (IleS+) andVL1970 (IleS17) were obtained. We tested the productivity of thesestrains as well as of the strain Rev874, culturing them as describedabove. The productivity was calculated as a ratio of valineconcentration in the culture medium to the optical density of thecultures. The results are presented in Table 7.

                  TABLE 7    ______________________________________                           Valine productivity    Strain tested IlsS allel                           g/l/OD560    ______________________________________    VL1968        ileS+.sup.                           0.01    VL1970        ileS17   0.76    Rev 874       ileS32   0.52    ______________________________________

It can be seen from Table 7 that ileS mutations markedly enhance valineproductivity of E. coli valine producers. Besides, mutation ileS17 whichis only partially suppressed by addition of isoleucine into the mediumhas a more pronounced effect on valine productivity. The more effectivevaline producer strain VL 1970 was constructed by combining in onebacterial genom mutation(s) in ilv operon, which have positive effectson valine production, with mutation ileS17, which impairs isoleucyl-tRNAsynthetase and can be suppressed only partially by addition of exogenousisoleucine into a medium.

4. Production of L-valine by the novel Escherichia coli strain VL1970.

E. coli VL1970 was cultivated at 37° C. for 18 hours in Erlenmeyerflasks with agitation (300 rpm) on the seed culture medium. It wasminimal medium M9 with glucose (1%). A main culture medium, containing30 g/l glucose, 5 g/l ammonium sulfate, 2 g/l K₂ HPO₄, 0.4 g/l MgSO₄·7H₂ O, O, 0.02 g/l FeSO₄ ·7H₂ O, 0.02 g/l MnSO₄ ·5H O, 0.6 g/l NaCl and0.25 g/l yeast autolysate, pH 7.2, was prepared.

300 ml of the medium was placed in a 0.5 liter jar-fermentor andsterilized at 121° C. for 15 minutes. The medium was inoculated with 30ml of the seed culture obtained above, and cultivated at 37° C. withstirring at 900 rotations per minute and introducing 1:1 volume of airper minute. The pH of the medium was maintained automatically around 7.2by introducing in the fermentor, on pH-monitor signal, the mixturecontaining a ratio of 6:1 50% glucose and 25% ammonia. The fermentationwas performed for 46 hours. By the end of the fermentation the culturemedium contained from 8.2 to 10.6 g/l of valine. This was 7 times morethan obtained by using an E. coli strain constructed by recombinant DNAtechniques.

The strain VL1970 has been deposited in the All-Union Collection ofIndustrial Microorganisms in VNIIGENETIKA(USSR) as VKPM B-4411.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A method of producing L-valine comprising:culturing an L-valine producing strain, wherein said strain comprises(a) a mutation affecting a valyl aminoacyl-tRNA synthetase therebyconferring cells with auxotrophy which can be partially suppressed,thereby allowing said cells to grow, by addition of valine into theculture medium; and (b) a mutation which destroys the negativeregulation of valine's biosynthesis, to yield a strain capable ofincreased production of valine, in nutrient media and crystallizingL-valine from the culture broth.
 2. A method of producing L-valinecomprising:culturing an Escherichia strain in nutrient media,accumulating L-valine in the media, and collecting L-valine from themedia, wherein said Escherichia strain is resistant to L-valine and hasa mutant gene selected from the group consisting of ileS2, ileS17 andileS32 instead of a wild-type ileS gene.